Novel sesquiterpenoid analogs

ABSTRACT

The present disclosure relates to novel sesquiterpenoid compounds as SHP2 and/or POLE3 inhibitors for potential treatment for cancers, and to methods of making and using the sesquiterpenoid compounds. The present invention therefore provides a method of using the disclosed compounds as chemosensitizations agent to a DNA damaging thugs for cancers.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefits of U.S. Provisional ApplicationSer. No. 62/714,785, filed Aug. 6, 2018, and 62/746,126, filed Oct. 16,2018, the contents of which are incorporated herein entirely.

GOVERNMENT RIGHTS

This invention was made with government support under National ScienceFoundation Career Award No. 1553820 awarded by National ScienceFoundation, and National Institutes of Health Award No. P30CA023168 andRO1 CA207288 awarded by National Institutes of Health. The United Statesgovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference a file68268-04_SEQ_ST25.txt including SEQ ID NO:1 to SEQ ID NO:4, provided ina computer readable form, created on Sep. 30, 2019 with a size of 2 KB,and filed with the present application. The sequence listing recorded inthe file is identical to the written sequence listing provided herein.

TECHNICAL FIELD

The present disclosure relates to novel sesquiterpenoid compounds asSHP2 and/or POLE3 inhibitors for potential treatment for cancers, and tomethods of making and using the sesquiterpenoid compounds.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Natural products have always held a privileged position as valuablesources and inspirations for new drug development and often lead to theidentification of new disease targets.

A family of Abies sesquiterpenoids represented by abiespiroside A,beshanzuenone C and beshanzuenone D have been reported. Abiespiroside Awas isolated from the Chinese fir tree species Abies delavayi and wasreported to possess potent inhibitory activity against the production ofnitric oxide, a therapeutic effect for inflammatory diseases such asarthritis. Beshanzuenones C and D were isolated from the shed tree barkof the critically endangered Chinese fir tree species Abiesbeshanzuensis.

Both beshanzuenone C and D were reported to inhibit PTP1B, a key targetfor the treatment of type-II diabetes and obesity with IC₅₀ values of59.7 and 40.4 μM, respectively. See Hu, C.-L., etc., European Journal ofOrganic Chemistry 2016, 10, 1832-1835.

Since rare and endangered plants have been shown to be superior sourcesfor drug compounds compared to other botanical sources, it is possiblethat these natural products and/or their analogs may inhibit otheradditional cellular targets and therefore possess new biologicalactivity for the treatment of other diseases.

SUMMARY

The present disclosure relates to novel sesquiterpenoid compounds asSHP2 and/or POLE3 inhibitors for potential treatment for cancers, and tomethods of making and using the sesquiterpenoid compounds.

In one embodiment, the present disclosure provides compounds of formulaI:

wherein

-   -   n is 1 or 2;    -   A is a C₃-C₈ saturated or unsaturated carbon ring;    -   R¹ and R² are each independently H, F, Cl, Br, optionally        substituted C₁-C₆ straight or branched alkyl or alkenyl,        optionally substituted aryl, or optionally substituted hetero        aryl comprising one or more O, N, or S;    -   R³ represents one or two optionally substituted C₁-C₄ straight        or branched alkyl; and    -   R⁴ represents one or more H, —OH, carbonyl group, optionally        substituted C₁-C₄ straight or branched alkyl, optionally        substituted C₁-C₄ straight or branched alkoxy, optionally        substituted aryl, or optionally substituted hetero aryl        comprising one or more O, N, or S.

In one embodiment, the present disclosure provides a method of usingcompounds disclosed in the present disclosure as SHP2 inhibitors.

In one embodiment, the present disclosure provides a method of usingcompounds disclosed in the present disclosure as POLE3 inhibitors.

In one embodiment, the present disclosure provides a method of usingcompounds disclosed in the present disclosure as SHP2 and/or POLE3inhibitors for cancer treatments.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodiments. Itwill nevertheless be understood that no limitation of the scope of thisdisclosure is thereby intended.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range.

In the present disclosure the term “substantially” can allow for adegree of variability in a value or range, for example, within 90%,within 95%, or within 99% of a stated value or of a stated limit of arange.

The term “substituted” as used herein refers to a functional group inwhich one or more hydrogen atoms contained therein are replaced by oneor more non-hydrogen atoms. The term “functional group” or “substituent”as used herein refers to a group that can be or is substituted onto amolecule. Examples of substituents or functional groups include, but arenot limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom ingroups such as hydroxyl groups, alkoxy groups, aryloxy groups,aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups includingcarboxylic acids, carboxylates, and carboxylate esters; a sulfur atom ingroups such as thiol groups, alkyl and aryl sulfide groups, sulfoxidegroups, sulfone groups, sulfonyl groups, and sulfonamide groups; anitrogen atom in groups such as amines, azides, hydroxylamines, cyano,nitro groups, N-oxides, hydrazides, and enamines; and other heteroatomsin various other groups.

Non-limiting examples of substituents, that can be bonded to asubstituted carbon (or other such as nitrogen) atom include F, Cl, Br,I, OR, OC(O)N(R)₂, CN, NO, NO₂, ONO₂, azido, CF₃, OCF₃, R, O (oxo), S(thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR,SO₂R, SO₂N(R)₂, SO₃R, (CH₂)₀₋₂P(O)OR₂, C(O)R, C(O)C(O)R, C(O)CH₂C(O)R,C(S)R, C(O)OR, OC(O)R, C(O)N(R)₂, OC(O)N(R)₂, C(S)N(R)₂,(CH₂)₀₋₂N(R)C(O)R, (CH₂)₀₋₂N(R)C(O)OR, (CH₂)₀₋₂N(R)N(R)₂, N(R)N(R)C(O)R,N(R)N(R)C(O)OR, N(R)N(R)CON(R)₂, N(R)SO₂R, N(R)SO₂N(R)₂, N(R)C(O)OR,N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R,C(═NH)N(R)₂, C(O)N(OR)R, or C(═NOR)R wherein R can be hydrogen or acarbon-based moiety, and wherein the carbon-based moiety can itself befurther substituted; for example, wherein R can be hydrogen, alkyl,acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, orheteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl,heterocyclyl, heteroaryl, or heteroarylalkyl or R can be independentlymono- or multi-substituted; or wherein two R groups bonded to a nitrogenatom or to adjacent nitrogen atoms can together with the nitrogen atomor atoms form a heterocyclyl, which can be mono- or independentlymulti-substituted.

The term “aryl” as used herein refers to substituted or unsubstitutedcyclic aromatic hydrocarbons that do not contain heteroatoms in thering. Thus aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl,anthracenyl, and naphthyl groups. In some embodiments, aryl groupscontain about 6 to about 14 carbons (C₆-C₁₄) or from 6 to 10 carbonatoms (C₆-C₁₀) in the ring portions of the groups. Aryl groups can beunsubstituted or substituted, as defined herein. Representativesubstituted aryl groups can be mono-substituted or substituted more thanonce, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substitutedphenyl or 2-8 substituted naphthyl groups, which can be substituted withcarbon or non-carbon groups such as those listed herein.

A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase“heterocyclyl group” includes fused ring species including those thatinclude fused aromatic and non-aromatic groups. Representativeheterocyclyl groups include, but are not limited to pyrrolidinyl,azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl,indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl,pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, triazyolyl, tetrazolyl,benzoxazolinyl, benzthiazolinyl, and benzimidazolinyl groups.

The terms “halo,” “halogen,” or “halide” group, as used herein, bythemselves or as part of another substituent, mean, unless otherwisestated, a fluorine, chlorine, bromine, or iodine atom. The compoundsdescribed herein may contain one or more chiral centers, or mayotherwise be capable of existing as multiple stereoisomers. It is to beunderstood that in one embodiment, the invention described herein is notlimited to any particular stereochemical requirement, and that thecompounds, and compositions, methods, uses, and medicaments that includethem may be optically pure, or may be any of a variety of stereoisomericmixtures, including racemic and other mixtures of enantiomers, othermixtures of diastereomers, and the like. It is also to be understoodthat such mixtures of stereoisomers may include a single stereochemicalconfiguration at one or more chiral centers, while including mixtures ofstereochemical configuration at one or more other chiral centers.

Similarly, the compounds described herein may include geometric centers,such as cis, trans, E, and Z double bonds. It is to be understood thatin another embodiment, the invention described herein is not limited toany particular geometric isomer requirement, and that the compounds, andcompositions, methods, uses, and medicaments that include them may bepure, or may be any of a variety of geometric isomer mixtures. It isalso to be understood that such mixtures of geometric isomers mayinclude a single configuration t one or more double bonds, whileincluding mixtures of geometry at one or more other double bonds.

The term “optionally substituted,” or “optional substituents,” as usedherein, means that the groups in question are either unsubstituted orsubstituted with one or more of the substituents specified. When thegroups in question are substituted with more than one substituent, thesubstituents may be the same or different. When using the terms“independently,” “independently are,” and “independently selected from”mean that the groups in question may be the same or different. Certainof the herein defined terms may occur more than once in the structure,and upon such occurrence each term shall be defined independently of theother.

In one embodiment, the present disclosure provides compounds of formulaI:

wherein

-   -   n is 1 or 2;    -   A is a C₃-C₈ saturated or unsaturated carbon ring;    -   R¹ and R² are each independently H, F, Cl, Br, optionally        substituted C₁-C₆ straight or branched alkyl or alkenyl,        optionally substituted aryl, or optionally substituted hetero        aryl comprising one or more O, N, or S;    -   R³ represents one or two optionally substituted C₁-C₄ straight        or branched alkyl; and    -   R⁴ represents one or more H, —OH, carbonyl group (═O),        optionally substituted C₁-C₄ straight or branched alkyl,        optionally substituted C₁-C₄ straight or branched alkoxy,        optionally substituted aryl, or optionally substituted hetero        aryl comprising one or more O, N, or S.

In one embodiment, the present disclosure provides compounds of formulaI, wherein A is a C₆ saturated or unsaturated carbon ring. In oneaspect, A is a C₆ unsaturated carbon ring.

In one embodiment, the present disclosure provides compounds of formulaI, wherein R¹ and R² are each independently H, F, Cl, Br, C₁-C₆ straightor branched alkyl or alkenyl, an aryl selected from phenyl, azulenyl,heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl,anthracenyl, or naphthyl; a hetero aryl selected from pyrrolidinyl,azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl,indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, or tetrahydrofuranyl.

In one embodiment, the present disclosure provides compounds of formulaI, wherein R¹ and R² are each independently H, F, Cl, Br, C₁-C₆ straightor branched alkyl.

In one embodiment, the present disclosure provides compounds of formulaI, wherein R¹ and R² are each independently H.

In one embodiment, the present disclosure provides compounds of formulaI, wherein n is 2.

In one embodiment, the present disclosure provides compounds of formulaI, wherein one or more hydrogen on R¹ and/or R² may be optionallysubstituted by —OH, —F, —Cl, —Br, —CN, —NC, —N₃, or C₁-C₄ alkoxy.

In one embodiment, the present disclosure provides compounds of formulaI, wherein R³ represents one or two C₁-C₄ straight or branched alkyl,and one or more hydrogen on the C₁-C₄ straight or branched alkyl may beoptionally substituted by one or more —OH, —F, —Cl, —Br, —CN, —NC, or—N₃, or C₁-C₄ alkoxy. In one aspect, R³ is methyl group.

In one embodiment, the present disclosure provides compounds of formulaI, wherein R⁴ represents one or more substituting groups selected fromthe group of —OH, carbonyl group, C₁-C₄ straight or branched alkyl,C₁-C₄ straight or branched alkoxy, or a combination thereof, wherein oneor more H on the C₁-C₄ straight or branched alkyl and/or the C₁-C₄straight or branched alkoxy is optionally substituted by one or more—OH, —F, —Cl, —Br, —CN, —NC, or —N₃.

In one embodiment, the present disclosure provides compounds of formulaII,

wherein either R¹-R⁴ are all H, or at least one of R¹-R⁴ is not H.

In one embodiment, the present disclosure provides compounds of formulaII, wherein

-   -   R¹ and R² are each independently H, F, Cl, Br, optionally        substituted C₁-C₆ straight or branched alkyl or alkenyl,        optionally substituted aryl, or optionally substituted hetero        aryl comprising one or more O, N, or S;    -   R³ represents optionally substituted C₁-C₄ straight or branched        alkyl; and    -   R⁴ represents H, optionally substituted C₁-C₄ straight or        branched alkyl, optionally substituted C₁-C₄ straight or        branched alkoxy, optionally substituted aryl, or optionally        substituted hetero aryl comprising one or more O, N, or S.

In one embodiment, the present disclosure provides compounds of formulaII, wherein R¹ and R² are each independently H, F, Cl, Br, C₁-C₆straight or branched alkyl or alkenyl, an aryl selected from phenyl,azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl,anthracenyl, or naphthyl; a hetero aryl selected from pyrrolidinyl,azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl,indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, or tetrahydrofuranyl.

In one embodiment, the present disclosure provides compounds of formulaII, wherein R¹ and R² are each independently H, F, Cl, Br, C₁-C₆straight or branched alkyl.

In one embodiment, the present disclosure provides compounds of formulaII, wherein R¹ and R² are each independently H.

In one embodiment, the present disclosure provides compounds of formulaII, wherein one or more hydrogen of R¹ and/or R² may be optionallysubstituted by —OH, —F, —Cl, —Br, —CN, —NC, —N₃, or C₁-C₄ alkoxy.

In one embodiment, the present disclosure provides compounds of formulaII, wherein R¹ and R² are H.

In one embodiment, the present disclosure provides compounds of formulaII, wherein R³ represents C₁-C₄ straight or branched alkyl, and one ormore hydrogen on the C₁-C₄ straight or branched alkyl may be optionallysubstituted by one or more —OH, —F, —Cl, —Br, —CN, —NC, or —N₃, or C₁-C₄alkoxy. In one aspect, R³ is methyl group.

In one embodiment, the present disclosure provides compounds of formulaII, wherein R⁴ represents C₁-C₄ straight or branched alkyl, or C₁-C₄straight or branched alkoxy, wherein one or more H on the C₁-C₄ straightor branched alkyl or the C₁-C₄ straight or branched alkoxy is optionallysubstituted by —OH, —F, —Cl, —Br, —CN, —NC, or —N₃.

In one embodiment, the present disclosure provides compounds of formulaIII,

wherein either R¹-R⁴ are all H, or at least one of R¹-R⁴ is not H.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein

-   -   R¹ and R² are each independently H, F, Cl, Br, optionally        substituted C₁-C₆ straight or branched alkyl or alkenyl,        optionally substituted aryl, or optionally substituted hetero        aryl comprising one or more O, N, or S;    -   R³ represents optionally substituted C₁-C₄ straight or branched        alkyl; and    -   R⁴ represents H, optionally substituted C₁-C₄ straight or        branched alkyl, optionally substituted C₁-C₄ straight or        branched alkoxy, optionally substituted aryl, or optionally        substituted hetero aryl comprising one or more O, N, or S.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein R¹ and R² are each independently H, F, Cl, Br, C₁-C₆straight or branched alkyl or alkenyl, an aryl selected from phenyl,azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl,anthracenyl, or naphthyl; a hetero aryl selected from pyrrolidinyl,azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl,indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, or tetrahydrofuranyl.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein R¹ and R² are each independently H, F, Cl, Br, C₁-C₆straight or branched alkyl.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein R¹ and R² are each independently H.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein one or more hydrogen of R¹ and/or R² may be optionallysubstituted by —OH, —F, —Cl, —Br, —CN, —NC, —N₃, or C₁-C₄ alkoxy.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein R³ represents C₁-C₄ straight or branched alkyl, and one ormore hydrogen on the C₁-C₄ straight or branched alkyl may be optionallysubstituted by one or more —OH, —F, —Cl, —Br, —CN, —NC, or —N₃, or C₁-C₄alkoxy. In one aspect, R³ is methyl group.

In one embodiment, the present disclosure provides compounds of formulaIII, wherein R⁴ represents C₁-C₄ straight or branched alkyl, or C₁-C₄straight or branched alkoxy, wherein one or more H on the C₁-C₄ straightor branched alkyl or the C₁-C₄ straight or branched alkoxy is optionallysubstituted by —OH, —F, —Cl, —Br, —CN, —NC, or —N₃

In one embodiment, the present disclosure provides compounds of formulaI, II or III, wherein the compounds are selected from the groupconsisting of:

or any stereoisomer such as an enantiomer or diastereomer, or acombination thereof.

In one embodiment, the present disclosure provides compounds of formulaI, II or III, wherein the compounds are selected from the groupconsisting of:

and any combination thereof.

In any embodiment, compounds of formula I, II, or III may be anystereoisomer such as an enantiomer or diastereomer, or a combinationthereof.

In one embodiment, the present invention provides a compound of FormulaI and/or II, and/or III as SHP2 inhibitor.

In one embodiment, the present invention provides a compound of FormulaI and/or IL and/or as a POLE3 inhibitor.

In one embodiment, the present invention provides a compound of FormulaI and/or II, and/or III as SHP2 and/or POLE3 inhibitors for cancertreatments.

In one embodiment, the present invention provides a method of using acompound of Formula I and/or II, and/or III as a chemosensitizationagent to a DNA damaging drugs for cancer. In one aspect, the DNAdamaging drug for cancer is etoposide.

Experiments

Reactions were performed using standard syringe techniques under argonunless stated otherwise. Starting materials and reagents were used asreceived from suppliers (Aldrich, Alfa Aeser, Acros.) Anhydrous THF wasdistilled over sodium benzophenone under argon. Acetonitrile (CH₃CN),dichloromethane (CH₂Cl₂), methanol (MeOH) and toluene were purified bypassing the previously degassed solvents through activated aluminacolumns. Flash chromatography was performed using silica gel (230-400mesh). Thin layer chromatography (TLC) was performed using glass-backedsilica plates (Silicycle). NMR spectra were recorded on a Bruker ARX-400spectrometer or AV-500 spectrometer at room temperature. Chemical shifts(in ppm) are given in reference to the solvent signal [¹H NMR: CDCl₃(7.26); ¹³C NMR: CDCl₃ (77.2)]. ¹H NMR data are reported as follows:chemical shifts (6 ppm), multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, quin=quintuplet, m=multiplet, br=broad), coupling constant(Hz), and integration. ¹³C NMR data are reported in terms of chemicalshift and multiplicity. IR data were recorded on a Thermo Nicolet Nexus470 FTIR. High-resolution mass measurements for compoundcharacterization were carried out using a FinniganMAT XL95 doublefocusing mass spectrometer system.

Preparation 1:(1R,2R,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol

The synthesis of Preparation 1 is illustrated with Scheme 1:

To a solution of compound A (15.0 g, 90.2 mmol, prepared in 48% yieldover 3 steps from (+)-carvone as described by Dos Santos et al., see DosSantos, R. B.; Brocksom, T. J.; Zanotto, P. R.; Brocksom, U. Molecules,2002, 7, 129-134) in THF (450 mL) was added sodium borohydride (6.83 g,180.5 mmol) at room temperature and the resulting solution was stirredfor 3 h. Water (200 mL) and ethyl acetate (200 mL) were added and theaqueous phase was extracted three times with ethyl acetate (3×100 mL.)The combined organic phases were washed with brine (200 mL), dried oversodium sulfate, filtered and concentrated under reduced pressure. Theresulting residue was purified by flash chromatography (hexanes:ethylacetate 2:1) to yield the desired preparation 1 as a white solid (13.1g, 80%) and side product S1 as a colorless oil (2.63 g, 16%). Spectraldata matched that reported by Koo et al. See Kim, H. J.; Su, L.; Jung,H.; Koo, S. Org. Lett. 2011, 13, 2682-2685.

Preparation 2:(4S,4aR,8R,8aR)-8-hydroxy-4,7-dimethyl-3,4,4a,5,8,8a-hexahydro-2H-chromen-2-one

The synthesis of Preparation 2 is illustrated with Scheme 2.

Preparation 3:(4S,4aR,8R,8aR)-8-((tert-butyldimethylsilyl)oxy)-4,7-dimethyl-3,4,4a,5,8,8a-hexahydro-2H-chromen-2-one

The synthesis of Preparation 3 is illustrated with Scheme 3:

To a stirred solution of a mixture of Preparation 2/S2 (7.20 g, 36.69mmol, 4:1 mixture) and imidazole (12.49 g, 183.4 mmol) in CH₂Cl₂ (183mL) was added tert-butyldimethylsilyl chloride (12.17 g, 80.72 mmol) inportions at 0° C. The resulting solution was warmed to room temperatureand stirred for 16 h. Water (50 mL) was added and the aqueous phase wasextracted with CH₂Cl₂ (3×20 mL.) The combined organic phases were washedwith brine (50 mL), dried over sodium sulfate, filtered and concentratedunder reduced pressure. The resulting yellow solid was purified by flashchromatography (hexanes:ethyl acetate 20:1) to yield 14 as a white solid(8.54 g, 75%.) and S2 as a white solid (1.48 g, 13%). Preparation 3(less polar isomer): ¹H NMR (500 MHz, CDCl₃) δ 5.41 (dq, J=5.4, 1.7 Hz,1H), 4.21-4.10 (m, 1H), 4.01 (dd, J=11.6, 7.6 Hz, 1H), 2.67 (dd, J=17.7,5.9 Hz, 1H), 2.38-2.19 (m, 1H), 2.11 (dd, J=17.7, 10.6 Hz, 1H),1.84-1.73 (m, 2H), 1.70 (s, 3H), 1.44 (qd, J=10.8, 5.2 Hz, 1H), 0.96 (d,J=6.5 Hz, 3H), 0.90 (s, 9H), 0.21 (s, 3H), 0.13 (s, 3H). ¹³C NMR (125MHz, CDCl₃) δ 170.2, 135.9, 121.8, 86.3, 75.1, 40.0, 38.1, 32.0, 28.9,26.2 (3C), 20.0, 18.9, 18.6, −3.6, −4.6. IR (neat, cm⁻¹): 2952, 2928,2857, 1717, 1381, 1248, 1074. HRMS (ESI): m/z=311.2042 calc. forC₁₇H₃₁O₃Si[M+H]⁺, found 311.2040. Compound S3 (more polar isomer): ¹HNMR (500 MHz, CDCl₃) δ 5.47-5.43 (m, 1H), 4.29-4.08 (m, 2H), 2.60 (dd,J=17.3, 5.7 Hz, 1H), 2.45 (dd, J=17.3, 3.1 Hz, 1H), 2.13-1.91 (m, 4H),1.72 (s, 3H), 1.02 (d, J=7.1 Hz, 3H), 0.93 (s, 9H), 0.24 (s, 3H), 0.15(s, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 170.4, 135.7, 122.4, 82.5, 76.1,39.0, 37.1, 28.5, 27.7, 26.3 (3C), 20.1, 18.7, 14.4, −3.5, −4.6. IR(neat, cm⁻¹) 2955, 2929, 2856, 1732, 1472, 1249, 1061. HRMS (ESI):m/z=311.2042 calc. for C₁₇H₃₁O₃Si[M+H]⁺, found 311.2040.

Preparation 4:(2R,4S,4aR,8R,8aR)-8-((tert-butyldimethylsilyl)oxy)-4,7-dimethyl-4′-methylene-3,3′,4,4a,4′,5,8,8a-octahydro-5′H-spiro[chromene-2,2′-furan]-5′-one

The synthesis of Preparation 4 is illustrated with Scheme 4:

To a stirred solution of Preparation 3 (4.38 g, 14.1 mmol) and activatedzinc dust (4.61 g, 70.5 mmol) in dioxane (14 mL) heated to 45° C. wasadded a solution of methyl 2-(bromomethyl)acrylate (4.22 g, 23.6 mmol,prepared as described by Ryu et al. See Kippo, T.; Fukuyama, T.; Ryu, I.Org. Lett. 2011, 13, 3864-3867.) in dioxane (14 mL). The mixture wasstirred at the same temperature for 16 h, then 2 N HCl (20 mL) and ethylacetate (20 mL) were added. The aqueous phase was extracted three timeswith ethyl acetate (10 mL) and the combined organic layers dried oversodium sulfate, filtered, and concentrated under reduced pressure. Theresulting residue was purified by flash chromatography (hexanes:ethylacetate 20:1) to yield Preparation 4 as a white solid (3.48 g, 65%.)¹HNMR (500 MHz, CDCl₃) δ 6.25 (dd, J=3.2, 2.4 Hz, 1H), 5.68-5.57 (m, 1H),5.40 (dq, J=5.1, 1.6 Hz, 1H), 4.04 (dq, J=7.6, 1.6 Hz, 1H), 3.79 (dd,J=11.4, 7.5 Hz, 1H), 2.96 (dt, J=17.3, 2.3 Hz, 1H), 2.81 (dt, J=17.3,3.0 Hz, 1H), 2.33-2.15 (m, 1H), 1.95-1.79 (m, 2H), 1.72 (ddq, J=11.8,5.9, 2.9 Hz, 1H), 1.67 (s, 3H), 1.54-1.40 (m, 1H), 1.29-1.17 (m, 1H),0.91 (d, J=6.3 Hz, 3H), 0.84 (s, 9H), 0.05 (s, 3H), 0.00 (s, 3H.)¹³C NMR(125 MHz, CDCl₃) δ 169.1, 135.8, 134.8, 122.7, 122.2, 105.0, 79.5, 74.9,43.0, 41.4, 40.9, 30.7, 28.6, 26.2 (3C), 20.4, 18.7, 18.5, −3.6, −4.2.IR (neat, cm⁻¹): 2949, 2929, 2882, 2857, 1768, 1250, 1123, 961, 838.HRMS (ESI): m/z=379.2305 calc. for C₂₁H₃₄O₄Si[M+H]⁺, found 379.2302.

EXAMPLES Example 1:(2R,4S,4aR,8R,8aR)-8-hydroxy-4,7-dimethyl-4′-methylene-3,3′,4,4a,4′,5,8,8a-octahydro-5′H-spiro[chromene-2,2′-furan]-5′-one

The synthesis of Example 1 is illustrated with Scheme 5:

Preparation 4 (1.50 g, 3.96 mmol) was dissolved in THF (13.2 mL) andhydrogen fluoride pyridine (˜70% hydrogen fluoride basis, 3.57 mL) wasadded dropwise at 0° C. The resulting solution was stirred for 16 h atroom temperature, then cooled to 0° C. and saturated sodium bicarbonatesolution (10 mL) was added dropwise, followed by ethyl acetate (10 mL).The aqueous phase was extracted three times with ethyl acetate (10 mL)and the organic phases were combined, washed with brine, dried oversodium sulfate, filtered and concentrated under reduced pressure. Theresulting residue was purified by flash chromatography (hexanes:ethylacetate 4:1 to 1:1) to yield Example 1 (1.01 g, 96%) as a white solid.¹H NMR (500 MHz, CDCl₃) δ 6.28 (dd, J=3.2, 2.5 Hz, 1H), 5.66 (t, J=2.5Hz, 1H), 5.40 (dq, J=5.7, 2.0 Hz, 1H), 4.04 (dd, J=7.6, 3.7 Hz, 1H),3.78 (dd, J=11.4, 8.0 Hz, 1H), 2.97 (dt, J=17.2, 2.3 Hz, 1H), 2.83 (dt,J=17.2, 2.9 Hz, 1H), 2.36-2.27 (m, 1H), 2.03 (d, J=3.5 Hz, 1H),1.96-1.84 (m, 2H), 1.74 (s, 3H), 1.54-1.46 (m, 1H), 1.31-1.21 (m, 1H),0.93 (d, J=6.4 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 169.0, 134.3, 123.1,122.2, 104.8, 79.39, 73.9, 42.6, 40.9, 40.7, 30.7, 28.8, 18.7, 18.5. IR(neat, cm⁻¹): 3477, 2969, 2954, 2887, 1760, 1215, 1131. HRMS (ESI):m/z=287.1260 calc. for C₁₅H₂₀O4[M+Na]⁺, found 287.1256.

Example 2:(2R,4S,4aR,8aR)-4,7-dimethyl-4′-methylene-3,3′,4,4a,4′,8a-hexahydro-5′H-spiro[chromene-2,2′-furan]-5′,8(5H)-dione

Example 3:(2R,4S,4aR,8aR)-7-(hydroxymethyl)-4-methyl-4′-methylene-3,3′,4,4a,4′,8a-hexahydro-5′H-spiro[chromene-2,2′-furan]-5′,8(5H)-dione

The synthesis of Example 2 and Example 3 is illustrated with Scheme 6:

A solution of Example 1 (63 mg, 0.239 mmol) and selenium dioxide (132mg, 1.19 mmol) in dioxane (4.0 mL) was stirred in a sealed vial at 80°C. for 2 h. After cooling to room temperature, the reaction mixture wasfiltered through Celite and concentrated under reduced pressure. Theresulting residue was purified by flash chromatography (ethyl acetate tohexanes 2:1 to separate Example 2 from Example 3 and Example 3′,followed by methylene chloride:methanol 95:5 to separate 30 from 31) toyield Example 2 as a white solid (35 mg, 56%), Example 3 as a whitesolid (4 mg, 6%), and Example 3′ as a white solid (15 mg, 22%). Example2: ¹H NMR (500 MHz, CDCl₃) δ 6.66-6.60 (m, 1H), 6.27 (ddd, J=5.1, 3.2,1.6 Hz, 1H), 5.68 (t, J=2.5 Hz, 1H), 4.40 (d, J=12.7 Hz, 1H), 3.18 (dt,J=17.4, 2.3 Hz, 1H), 2.83 (dt, J=17.4, 3.0 Hz, 1H), 2.66 (dtt, J=18.7,4.7, 1.4 Hz, 1H), 2.16-2.05 (m, 2H), 1.93 (dd, J=13.8, 3.9 Hz, 1H), 1.78(s, 3H), 1.70 (ddt, J=12.8, 10.7, 5.3 Hz, 1H), 1.52 (dd, J=13.8, 7.9 Hz,1H), 0.98 (d, J=6.5 Hz, 3H.)¹³C NMR (125 MHz, CDCl₃) δ 195.4, 168.9,142.4, 135.0, 133.8, 123.7, 104.9, 77.7, 44.3, 42.1, 40.5, 31.2, 29.2,18.2, 15.8. IR (neat, cm⁻¹) 2924, 1768, 1690, 1227, 1123. m/z=263.1283calc. for C₁₅H₁₈O₄[M+H]⁺, found 263.1277. IR (neat, cm⁻¹): 2924, 1768,1690, 1227, 1013. HRMS (ESI): m/z=263.1283 calc. for C₁₅H₁₈O₄[M+H]⁺,found 263.1283. Example 3: ¹H NMR (500 MHz, CDCl₃) δ 6.89-6.85 (m, 1H),6.30 (dd, J=3.2, 2.4 Hz, 1H), 5.70 (dd, J=2.8, 2.1 Hz, 1H), 4.46 (d,J=12.8 Hz, 1H), 4.31 (d, J=13.2 Hz, 1H), 4.22 (dd, J=13.6, 5.5 Hz, 1H),3.17 (dt, J=17.4, 2.3 Hz, 1H), 2.84 (dt, J=17.4, 3.0 Hz, 1H), 2.77(dddt, J=19.0, 5.8, 4.8, 1.0 Hz, 1H), 2.30 (t, J=6.6 Hz, 1H), 2.23-2.10(m, 2H), 1.96 (dd, J=13.8, 3.9 Hz, 1H), 1.80-1.70 (m, 1H), 1.55 (dd,J=13.8, 12.5 Hz, 1H), 1.00 (d, J=6.6 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ195.9, 168.8, 144.0, 137.6, 133.6, 123.9, 104.8, 77.51, 61.6, 44.1,42.1, 40.4, 31.2, 29.1, 18.2. IR (neat, cm⁻¹): 3379, 2923, 2853, 1769,1561, 1412, 1110. HRMS (ESI): m/z=279.1232 calc. for C₁₅H₁₈O₄[M+H]⁺,found 279.1217. Example 3′: ¹H NMR (500 MHz, CDCl₃) δ 6.29-6.28 (m, 1H),5.68-5.66 (m, 2H), 4.34-4.32 (m, 1H), 4.17 (s, 2H), 3.84 (dd, J=11.4,8.0 Hz, 1H), 2.99-2.94 (dt, J=15.0, 2.3 Hz, 1H), 2.86-2.80 (dt, J=17.2,3.0 Hz, 1H), 2.41 (dt, J=18.0, 5.4 Hz, 1H), 1.95 (m, 1H), 1.92 (m, 1H),1.81 (m, 1H), 1.56-1.48 (m, 1H), 1.32-1.22 (m, 1H), 0.94 (d, J=6.4 Hz,3H). ¹³C NMR (125 MHz, CDCl₃) δ 168.9, 136.6, 134.1, 124.8, 123.1,104.6, 78.7, 73.0, 64.8, 42.4, 40.7, 40.1, 30.5, 28.6, 18.4. IR (neat,cm⁻¹): 3420, 2923, 1764, 1216. HRMS (ESI) m/z=303.1203 calc. forC₁₅H₂₀O₅Na [M+Na]⁺=303.1203, found 303.1203.

Example 4:(2R,4S,4aR,8aR)-7-(azidomethyl)-4-methyl-4′-methylene-3,3′,4,4a,4′,8a-hexahydro-5′H-spiro[chromene-2,2′-furan]-5′,8(5H)-dione

The synthesis of Example 4 is illustrated with Scheme 7

To a solution of Example 3 (10 mg, 0.036 mmol) in CH₂Cl₂ (0.36 mL) wasadded Et₃N (6 μL, 0.0432 mmol). The resulting solution was cooled to 0°C. and methanesulfonyl chloride (2.5 μL, 0.0432 mmol) was added. Afterstirring for 1 h, brine (1 mL) and CH₂Cl₂ (1 mL) were added. The aqueousphase was extracted with CH₂Cl₂ (3×1 mL) and the combined organic layerswere dried over sodium sulfate, filtered and concentrated under reducedpressure. The resulting crude residue was dissolved in DMF (0.36 mL) andsodium azide (11.7 mg, 0.18 mmol) was added. The reaction vessel wascovered with aluminium foil and the solution stirred for 18 h. Water (1mL) and ethyl acetate (1 mL) were added and the aqueous phase extractedwith ethyl acetate (3×1 mL.) The combined organic phases were washedwith brine (2 mL), dried over sodium sulfate, filtered and concentratedunder reduced pressure. The crude residue was purified by flashchromatography (methylene chloride:ethyl acetate 50:1 to 20:1 to 10:1)to yield Example 4 as a colorless oil (3.8 mg, 35%). ¹H NMR (500 MHz,CDCl₃) δ 6.91 (d, J=6.2 Hz, 1H), 6.30 (t, J=2.9 Hz, 1H), 5.70 (t, J=2.5Hz, 1H), 4.47 (d, J=12.8 Hz, 1H), 3.99 (q, J=14.5 Hz, 2H), 3.18 (dt,J=17.4, 2.3 Hz, 1H), 2.89-2.76 (m, 2H), 2.27-2.11 (m, 2H), 1.96 (dd,J=13.8, 3.9 Hz, 1H), 1.80-1.69 (m, 1H), 1.01 (d, J=6.5 Hz, 3H). ¹³C NMR(125 MHz, CDCl₃) δ 193.9, 168.8, 145.2, 133.6, 123.9, 104.7, 77.4, 49.4,44.0, 42.0, 40.4, 31.3, 29.2, 18.2. IR (neat, cm⁻¹): 2924, 2853, 2105,1770, 1634, 1261, 1014. HRMS (ESI): m/z=326.1117 calc. forC₁₅H₁₉N₃O₄[M+Na]⁺, found 326.1110.

Target Identification Procedure

Materials and General Methods

DMEM/High glucose media with GlutaMAX and sodium pyruvate, phosphatebuffered saline (PBS), MEM Non-Essential Amino Acids, PenicillinStreptomycin (Pen/Strep), Trypsin-EDTA and OptiMEM were obtained fromLife Technologies. Protein concentration was determined using theBradford assay (Bio-Rad). Cloning of POLE3-GFP was done using theGateway technology (Thermo Fisher Scientific). Plasmids were preparedusing the QIAprep Spin Miniprep Kit (Qiagen). For all PCR reactions, Q5®High-Fidelity DNA Polymerase (New England BioLabs Inc) was used asrecommended by the manufacturer. PCR cleanup was performed using agarosegel electrophoresis with subsequent cutting of amplified bands usingQIAquick Gel Extraction Kit (Qiagen) following the protocol of theproducer. For the BP and the LR reactions the Gateway® BP Clonase®Enzyme Mix and the Gateway® LR Clonase® Enzyme mix (Thermo FisherScientific) were used according to the protocol of the manufacturer.siRNA for POLE3 was purchased from Dharmacon (ON-TARGETplus HumanPOLE3—SMARTpool).

Cell Culture and Preparation of Lysates

MDA-MB-231, HeLa and 293T cells were maintained in DMEM media. All mediawere supplemented with 10% fetal calf serum (FCS), non-essential aminoacids and penicillin/streptomycin. Cells were grown at 37° C. under 5%CO₂ atmosphere. Cells were allowed to grow to confluence and wereharvested by scraping, centrifuged at 1′500×g for five min. at 4° C. andresuspended in PBS. Cells were lysed by sonication to form cell lysatesand protein concentration was determined using the Bradford assay.

Preparation of Proteomes for SDS-PAGE Experiments

MDA-MB-231 lysate (2 mg/mL, 25 μL) was treated with indicatedconcentrations of azide probes (Example 4) (1 μL of 25× stock in DMSO)for one hour at r.t. Click chemistry was initiated by the addition ofTAMRA alkyne (Sigma-Aldrich, 30 μM, 25× stock in DMSO),tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Alfa Aesar, 1 mM,fresh 50× stock in water),tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Sigma-Aldrich,100 μM, 16× stock in DMSO:tBuOH 1:4), and copper(II) sulfate (1 mM, 50×stock in water) to the lysate and incubated in the dark for one hour atr.t. SDS-PAGE reducing loading buffer (4×) was added and proteins wereseparated using a 10% SDS-PAGE gel. Gels were visualized using an AzureBiosystems Sapphire Biomolecular Imager, then stained using Coomassie.Images were quantified with ImageJ (V1.51).

Gel-Based In Vitro JW-RF-001 Competition of Labeling with Example 4

MDA-MB-231 lysate (2 mg/mL, 25 μL) was treated with either 3 mMJW-RF-001 (1 μL of 25× stock in DMSO) or DMSO for one hour at r.t.Subsequently, lysate was incubated with 30 μM Example 4 (1 μL, of 25×stock in DMSO) for one hour at r.t. Click chemistry, reducing SDS-PAGEand visualization were performed as described above. The study showedthat Example 4 binded to cysteines, as pretreatment of the lysates withthe cysteine-reactive reagent JW-RF-001 completely abolished thelabeling.

Cloning and Site-Directed Mutagenesis

The POLE3-WT gene was amplified from a cDNA library derived from HeLacells and flanked with Attb-sites using the following primer: forward5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAT GGC GGA GAG GCC C-3′ (SEQID NO:1) and reverse 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC GTT GTCTAC TTC TTC CTC TTC ATT CT-3′ (SEQ ID NO:2). The PCR reaction wascleaned up and the PCR product recombined into the pDONR 221. pENTR wasused for transferring the POLE3-WT gene into the pcDNA 6.2 N-EmGFP-DESTvia the LR reaction to merge the emGFP gene to the 3′ of the gene toobtain POLE3-GFP-pEXP. This pEXP was used for PCR based site directedmutagenesis to generate the mutated pEXP vector POLE3-051S-GFP-pEXP(forward primer: 5′-TCC TCT GCT AAC AAC TTT GCA ATG AAA GGA-3′(SEQ IDNO:3); reverse primer: 5′-AGC AGA GGA TGT GGC GTA CAG CAC GAA GAC-3′(SEQ ID NO:4)). Afterwards the PCR reaction was incubated with DpnI (NewEngland BioLabs Inc, 37° C., 12 hours followed by 80° C. for 20 min.) todegrade POLE3-WT-GFP-pEXP. The pENTR and pEXP plasmids were propagatedvia transformation into E. coli DH5a using heat shock.

Transfection, Gel-Based In Vitro Labeling Experiments Using ProbeExample 4 and Western Blots

293T cells in a 3.5 cm Petri dish were transfected withPOLE3-WT-GFP-pEXP or POLE3-051S-GFP-pEXP using PEI-MAX. After 24 hours,lysates were prepared as described above. Proteins (1 mg/mL, 25 μL) wereincubated with indicated compounds at indicated concentrations (25×stock in DMSO) for three hours at 37° C. Subsequently, 30 μM Example 4(25× stock in DMSO) was added for one hour at r.t. Click chemistry,reducing SDS-PAGE and visualization were performed as described above.Proteins were transferred to a PVDF membrane using a Trans-Blot Turbotransfer system (Bio-Rad). Membrane was blocked with 3% BSA inTris-Buffered Saline with 0.1% Tween 20 (TBST) for one hour at r.t.Mouse monoclonal antibody against GFP (Roche, 1/1000 in 3% BSA in TBST)was added and incubated overnight at 4° C. Membrane was washed threetimes for five min. with TBST and incubated with anti-mouse Alexa Fluor647 (Jackson ImmunoResearch Inc., 1/500 in 3% BSA in TBST) for one hourat r.t. Membrane was washed four times for five min. with TBST andvisualized with an Azure Biosystems Sapphire Biomolecular Imager.

Gel-Based Determination of Kinetic Parameters K₁ and k_(inact)

WT POLES-GFP was overexpressed in 293T cells as described above andlysate was treated with indicated concentrations of Example 2 (1 μL of25× stock in DMSO) for 1.5, 2, 2.5, 3, 3.5 or 4 hours at 37° C. Sampleswere then treated with 30 μM Example 4 (1 μL of 25× stock in DMSO) forone hour at r.t. Click chemistry, reducing SDS-PAGE and visualizationwere performed as described above. Images were quantified using ImageJand the kinetic parameters were determined using GraphPad Prism(V7.02).). The application of Kitz and Wilson method (see Kitz, R.;Wilson, I. B. J. Biol. Chem. 1962, 237, 3245-3249) provided the Example2 POLE3-GFP binding constants K_(i) (56.9 μM) and k_(inact) (0.27min⁻¹), respectively.

To understand whether binding of Example 2 to C₅₁ of POLE3 is productiveand leads to the loss of function of this protein, POLE3 was knockeddown in HeLa cells using siRNA and compared by global proteomics theprotein expression profile in these cells versus cells treated withcompound Example 2 or DMSO as control. There are 224 overexpressed and216 downregulated proteins found in POLE3 KD cells versus cells treatedwith DMSO. Moreover, there is a significant overlap in up- anddownregulated proteins between the POLE3 KD cells and the cells after 6h treatment with Example 2. It indicated that binding by Example 2indeed leads to the loss of function of POLE3. It was found severalproteins involved in DNA repair that were upregulated in both POLE3 KDand Example 2-treated cells, such as DNA ligase 3, GEMIN2, PEA15 andGIT2. Possibly as a compensatory effect, we also observed fourfoldupregulation of POLE, another subunit of DNA polymerase ε. These resultssuggest that POLE3 may indeed be involved in endogenous DNA repairpathways, although clearly deeper biological insight is required todecipher the mechanistic role of POLE3 in these critical processes.

Visualization of DNA Double Strand Breaks for Microscopy (γH2AX)

HeLa cells were seeded (250′000 cells/mL) in a 6-well plate and reversetransfected with scrambled siRNA or POLE3 siRNA using RNAiMAX for twodays. Cells were trypsinizned, seeded (150′000 cells/mL) in a 24-wellplate containing a coverslip and left overnight to attach and grow.Cells were incubated for 6 hours in media without FCS with either DMSO(0.2% final), 30 μM etoposide, 30 μM Example 2 or 30 μM etoposidetogether with 30 μM Example 2. Cells were washed one time with PBS andfixed with methanol at −20° C. for 10 min. Cells were washed again twotimes with PBS and coverslip was transferred to a wet box.

Blocking was performed with a 5% bovine serum albumin (BSA) solution inPBS for 30 min. Cells were washed one time with PBS 0.1% BSA and rabbitantibody directed against H2AX pSer¹³⁹ (Sigma Aldrich H5912, 1/250 inPBS 0.1% BSA) was added and incubated for one hour at r.t. Cells werewashed three times with PBS 0.1% BSA and incubated for one hour withanti-rabbit IgG Alexa Fluor 488 (Jackson, 1/200 in PBS 0.1% BSA). Cellswere washed three times with PBS 0.1% BSA, one time with PBS, one timewith water and then mounted on a slide with ProLong Gold antifademountant with DAPI (Life Technologies). Cells were visualized with anLSM 700 confocal microscope (Zeiss).

Due to its potential involvement in DNA repair pathways, studies werecarried out to investigate the effect of POLE3 knockdown or inactivationby Example 2 in combination with a DNA damaging agent such as etoposide.Etoposide is a clinically applied drug to treat numerous types ofcancer. It forms a ternary complex with DNA and topoisomerase II toeventually cause DNA strand breakage. Briefly, HeLa cells were treatedor not with POLE3 siRNA and then subsequently exposed to treatment by 30μM etoposide, 30 μM Example 2, or 30 μM etoposide combined with 30 μMExample 2. Induced DNA damage was measured by confocal microscopy usingγH2AX staining as marker. Treatment with Example 2 alone did not lead toan increase in γH2AX signal intensity. However, intriguingly,significantly stronger γH2AX signal was observed in cells treated withboth etoposide and Example 2 versus etoposide only-treated cells.Likewise, POLE3 KD cells did not show increased DNA damage, but addingetoposide again caused increased γH2AX signal in comparison to theetoposide-treated wild type cells. These results demonstrate thattargeting POLE3 with small molecules may indeed be a novel strategy forchemosensitization to DNA damaging drugs in cancer.

Global Proteomics Profiling Using LC-MS/MS

Knockdown of POLE3 was achieved using RNAiMAx (Invitrogen) and POLE3siRNA (Dharmacon) via reverse transfection according to the manual ofthe manufacturer. Briefly, 9 μL of 20 μM siRNA was mixed with 9 μL ofRNAiMax reagent in 500 μL OptiMEM and incubated for 20 min. at r.t. HeLacells (250′000 cells/mL) were seeded in a 6-well plate on top andincubated for 48 hours at 37° C. Cells were washed twice with PBS andtreated with DMSO (0.1% final) or 30 μM Example 2 for 6 hours at 37° C.Cells were washed with PBS and lysate was prepared as mentioned above.To 25 μL lysate (1 mg/mL) in PBS, Urea was added to 6 M finalconcentration and incubated with 10 mM TCEP for 20 min. at r.t. underrotation. For alkylation, 25 mM IAA was added and incubated for 20 min.at r.t. under rotation. Solution was diluted to 2 M Urea with 50 mMNH₄HCO₃ and 1 mM CaCl₂, then 0.5 μg trypsin (Thermo Scientific) wasadded. Tryptic digestion was performed overnight at 37° C. Peptides weredesalted over a self-packed C18 spin column and dried. Samples wereanalyzed by LC-MS/MS (see below) and the MS data was processed withMaxQuant (see below).

In Situ Competitive Experiment for Mass Spectrometry

MDA-MB-231 or HeLa cells were seeded in 14 cm Petri dishes and left toattach and grow until confluence. Cells were washed one time with PBSand treated with either DMSO or 30 μM Example 2 for 6 hours in mediawithout FCS at 37° C. Lysate was prepared as described above. Lysate (2mg/mL, 0.75 mL) was treated with 30 μM Example 4 (100× stock in DMSO)for one hour at r.t. and then subjected to click chemistry.Photocleavable (PC) biotin alkyne (Click Chemistry Tools, 60 μM, 50×stock in DMSO), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1mM, 50× fresh stock in water),tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (100 μM, 16×stock in DMSO:tButanol 1:4), and copper(II) sulfate (1 mM, 50× stock inwater) were added to the proteome and left to react for one hour at r.t.Protein was precipitated by adding MeOH (4 vol.), CHCl₃ (1 vol.) andwater (3 vol.) to the reaction mixture and the turbid mixture wascentrifuged for five min. at 20′000×g at 4° C. yielding a protein layerbetween the aqueous and organic layers. The protein layer was isolated,dried and solubilized in 2% SDS in PBS via sonication. Tube wascentrifuged at 4′700×g for five min. and soluble fraction wastransferred to a new tube. PBS was added to give a final SDSconcentration of 0.2%. 160 μL of streptavidin agarose beads (ProteoChem)were added and the mixture was rotated for four hours at r.t. Beads werewashed with 1% SDS in PBS (1×10 mL), PBS (3×10 mL), and water (3×10 mL).Beads were resuspended in 6 M urea in PBS (500 μL), reduced with 10 mMneutralized TCEP (20× fresh stock in water) for 30 min. at r.t., andalkylated with 25 mM iodoacetamide (400 mM fresh stock in water) for 30min. at r.t. in the dark. Beads were pelleted by centrifugation(1′400×g, two min.) and resuspended in 150 μL of 2 M Urea, 1 mM CaCl₂(100× stock in water) and trypsin (Thermo Scientific, 1.5 μL of 0.5μg/μL) in 50 mM NH₄HCO₃. The digestion was performed for 6 hours at 37°C. Samples were acidified to a final concentration of 5% acetic acid,desalted over a self-packed C18 spin column and dried. Samples wereanalyzed by LC-MS/MS (see below) and the MS data was processed withMaxQuant (see below).

LC-MS/MS Analysis

Peptides were resuspended in water with 0.1% formic acid (FA) andanalyzed using EASY-nLC 1200 nano-UHPLC coupled to Q Exactive HF-XQuadrupole-Orbitrap mass spectrometer (Thermo Scientific). Thechromatography column consisted of a 30 cm long, 75 μm i.d.microcapillary capped by a 5 μm tip and packed with ReproSil-Pur 120C18-AQ 2.4 μm beads (Dr. Maisch GmbH). LC solvents were 0.1% FA in H₂O(Buffer A) and 0.1% FA in 90% MeCN: 10% H₂O (Buffer B). Peptides wereeluted into the mass spectrometer at a flow rate of 300 nL/min. over a240 min. linear gradient (5-35% Buffer B) at 65° C. Data was acquired indata-dependent mode (top-20, NCE 28, R=7′500) after full MS scan(R=60′000, m/z 400-1′300). Dynamic exclusion was set to 10 s, peptidematch to prefer and isotope exclusion was enabled.

MaxQuant Analysis

The MS data was analyzed with MaxQuant (V1.6.1.0) and searched againstthe human proteome (Uniprot) and a common list of contaminants (includedin MaxQuant). The first peptide search tolerance was set at 20 ppm, 10ppm was used for the main peptide search and fragment mass tolerance wasset to 0.02 Da. The false discovery rate for peptides, proteins andsites identification was set to 1%. The minimum peptide length was setto 6 amino acids and peptide re-quantification, label-freequantification (MaxLFQ) and “match between runs” were enabled. Theminimal number of peptides per protein was set to two. Methionineoxidation was searched as a variable modification andcarbamidomethylation of cysteines was searched as a fixed modification.

Protein Tyrosine Phosphatase Inhibition Evaluation

PTP Inhibition Study. Abies sesquiterpenoids and their analogs wereevaluated for their ability to inhibit the reaction catalyzed by PTP1Band SHP2 using p-nitrophenyl phosphatae (pNPP) as a substrate at pH 7and 25° C. The reaction was started in a 384 plate, by the addition of25 μL of the enzyme (40 nM stock concentration or 17.7 mg/ml) to 25 μLof reaction mixture containing 6 mM (2× the K_(m) value) of pNPP andvarious concentrations of the inhibitor (added by Echo, volume between2.5 to 500 nl), in a buffer containing 50 mM 3,3-dimethylglutarate, 1 mMEDTA, and ionic strength of 150 mM adjusted with NaCl. the initial rateat a series of pNPP concentrations was measured by following theproduction of p-nitrophenol. The reaction was quenched after 3 (PTP1B)or 15 (SHP2) min by the addition of 25 μl of 5N NaOH. The absorbance at405 nm was detected by a Spectra MAX 384 PLUS microplatespectrophotometer (Molecular Devices). IC₅₀ values were calculated byfitting the absorbance at 405 nm versus inhibitor concentration to thefollowing equation:

A ₁ /A ₀ =IC ₅₀/(IC ₅₀+[I])

wherein A₁ is the absorbance at 405 nm of the sample in the presence ofinhibitor; A₀ is the absorbance at 405 nm in the absence of inhibitor;and [I] is the concentration of the inhibitor.

Kinetic Characterization of SHP2 Inactivation by Example 3′. PTPinactivation by the vinyl sulfonates and sulfones was studied at 25° C.in a pH 7 buffer containing 50 mM sodium succinate, 1 mM EDTA, and ionicstrength of 150 mM adjusted with NaCl. The assay was performed in a 96well plate, starting with preparation of a series of inactivatorsolutions at various concentrations in one column. At appropriate timeintervals, aliquots of 4 μl were removed from the reaction and addedinto a 200 μl solution containing 20 mM pNPP at 25° C. in the abovebuffer. The remaining PTP activity was measured using proceduresdescribed above. The kinetic parameters of the inactivation reactionwere obtained by fitting the data to the following equations:

$\frac{A_{t}}{A_{0}} = {\frac{A_{\infty}}{A_{0}} - {\left( \frac{A_{0} - A_{\infty}}{A_{0}} \right)e^{{- k_{obs}} \cdot t}}}$

Example 2, Example 3, and Example 3′ provided SHP2 IC₅₀ values of44.9±9.6 and 3.3±0.1 respectively. Example 2, Example 3, and Example 3′also provided 10-fold preference for SHP2 over PTP1B. Therefore, theexemplified compounds in the present disclosure may be used as SHP2inhibitors for a variety of cancer treatments. See Frankson, R.; Yu,Z.-H.; Bai, Y.; Li, Q.; Zhang, R.-Y.; Zhang, Z.-Y. Cancer Res. 2017, 77,5701-5705.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

We claim:
 1. A compound of formula I:


2. The compound of claim 1, wherein the compound is used as an SHP2inhibitor for cancer treatments.